Protein kinase A (PKA) is a ubiquitous serine/threonine protein kinase involved in cell signaling by phosphorylating intracellular protein substrates in response to the secondary messenger, cyclic adenosine monophosphate (cAMP). The PKA holoenzyme is composed of two catalytic (C-) and two regulatory (R-) subunits that form an inactive tetramer. Upon the binding of cAMP to the R-subunits (each having two cAMP-binding sites), the tetramer dissociates into a dimer of R-subunits and two catalytically active C-subunits, thus enabling the phosphorylation of downstream PKA substrates.
The R-subunit has two major roles. First, it prevents access to the substrate-binding site on the C-subunit (in the absence of cAMP), and second, it determines subcellular localization of this kinase via specific interactions with A-kinase anchoring proteins (AKAPs). There are four isoforms of the PKA regulatory subunit (RIα, RIβ, RIIα, and RIIβ) that differ in their abundance, affinity for the PKA catalytic subunit, sensitivity to cAMP, and specificity for different AKAPs. This diversity determines cell-specific PKA localization and diversifies PKA-mediated signaling [Feliciello, A., et al., (2001), The biological functions of A-kinase anchor proteins, J Mol Biol 308, 99-114., Skalhegg, B. S., and Tasken, K. (2000), Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front Biosci 5, D678-693].
PKA is implicated in several cancers including endocrine (e.g. carney complex [Casey, M., et al., (2000), Mutations in the protein kinase A R1alpha regulatory subunit cause familial cardiac myxomas and Carney complex. J Clin Invest 106, R31-38., Kirschner, L. S. et al., (2000), Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex. Nat Genet. 26, 89-92] and pituitary [Lania, A. G., et al., (2004), Proliferation of transformed somatotroph cells related to low or absent expression of protein kinase a regulatory subunit 1A protein, Cancer Res 64, 9193-9198]) and non-endocrine (e.g. breast [Taimi, M., et al., (2001), Cyclic AMP-dependent protein kinase isoenzymes in human myeloid leukemia (HL60) and breast tumor (MCF-7) cells, Arch Biochem Biophys 392, 137-144., Miller, W. R. (2002), Regulatory subunits of PKA and breast cancer, Ann N Y Acad Sci 968, 37-48]). Additionally, PKA is suggested to be a therapeutic target for diseases of the immune system (e.g. SLE and HIV) [Skalhegg, et al., (2005), Protein kinase A (PKA)—a potential target for therapeutic intervention of dysfunctional immune cells, Curr Drug Targets 6, 655-664]. However, given the ubiquitous nature of this protein kinase, targeting the ATP-binding site of the catalytic subunit, would likely kill healthy cells.
Recently, an alternative strategy was used to target PKA in disease. Several studies have shown that disease progression in some cancers is correlated with abnormally high levels of the RIα isoform [Taimi, M., et al., (2001), Cyclic AMP-dependent protein kinase isoenzymes in human myeloid leukemia (HL60) and breast tumor (MCF-7) cells. Arch Biochem Biophys 392, 137-144., Miller, W. R. (2002). Regulatory subunits of PKA and breast cancer. Ann N Y Acad Sci 968, 37-48], and it is presumably the abundance of type Iα PKA holoenzyme (PKA-Iα) that causes aberrant phosphorylation and cancer. As a result, RIα antisense therapy is currently undergoing phase I/II clinical trials for the treatment of patients with malignant solid tumors [Nesterova, M. V., and Cho-Chung, Y. S., (2004), Antisense protein kinase A RIαalpha inhibits 7,12-dimethylbenz(a)anthracene-induction of mammary cancer: blockade at the initial phase of carcinogenesis, Clin Cancer Res 10, 4568-4577]. A small molecule that selectively inhibits the catalytic function of PKA-Iα holoenzyme would mimic the effect of RIα antisense therapy by repressing aberrant phosphorylation. In addition, a PKA-Iα antagonist has been suggested as a therapeutic agent to improve T cell responsiveness to HIV [Skalhegg, B. S., et al., (2005), Protein kinase A (PKA)—a potential target for therapeutic intervention of dysfunctional immune cells, Curr Drug Targets 6, 655-664]. On the other hand, a small molecule that selectively activates PKA may also be of benefit. For example, S49 T-lymphoma cells have been reported to undergo cAMP stimulated apoptosis through activation of PKA [Zhang, L., and Insel, P. A., (2004), The pro-apoptotic protein Bim is a convergence point for cAMP/protein kinase A- and glucocorticoid-promoted apoptosis of lymphoid cells, J Biol Chem 279, 20858-20865].
PKA Small Molecule Ligands
Two classes of small molecule effectors targeting PKA have been described. The first are ATP competitive that inhibit the catalytic function via interaction with the ATP-binding site of the C-subunit. However, ATP-competitors, such as H89 [Chijiwa, T., et al., (1990), Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89), of PC 12D pheochromocytoma cells. J Biol Chem 265, 5267-5272] and balanol [Akamine, P., et al., (2004), Balanol analogues probe specificity determinants and the conformational malleability of the cyclic 3′,5′-adenosine monophosphate-dependent protein kinase catalytic subunit, Biochemistry 43, 85-96] inhibit other protein kinases and non-related cellular receptors [Son, Y. K., et al., (2006), Direct inhibition of a PKA inhibitor, H-89 on KV channels in rabbit coronary arterial smooth muscle cells, Biochem Biophys Res Commun 341, 931-937].
The second class comprises cAMP competitors [Schwede, F., et al., (2000), Cyclic nucleotide analogs as biochemical tools and prospective drugs. Pharmacol Ther 87, 199-226]. Activation of the holoenzyme is induced by binding of four cAMP molecules, two per R-subunit. Cell permeable derivatives of cAMP act as agonists (activating the holoenzyme in the same way as cAMP itself) or antagonists (competing with cAMP for binding to the R-subunits but not activating the holoenzyme). However, the major drawback with this class of small molecules are their poor affinity for the PKA regulatory subunits (micromolar) leading to poor cellular responses.
Targeting the cAMP Binding Sites
The cAMP binding sites are targets for novel non-ATP competitive effectors of PKA function. To date, only cyclic nucleotide analogs have been reported to bind to these sites. However, as the cyclic phosphate group is a strict requirement for binding, these compounds exhibit poor in-vivo efficacy. Crystal structures for the free R-subunits [Wu, J., Brown, et al., (2004), RIα subunit of PKA: a cAMP-free structure reveals a hydrophobic capping mechanism for docking cAMP into site B. Structure 12, 1057-1065] as well as R-subunit bound to cAMP analogs [Wu, J., et al., (2004), Crystal structures of RIalpha subunit of cyclic adenosine 5′-monophosphate (cAMP)-dependent protein kinase complexed with (Rp)-adenosine 3′,5′-cyclic monophosphothioate and (Sp)-adenosine 3′,5′-cyclic monophosphothioate, the phosphothioate analogues of cAMP, Biochemistry 43, 6620-6629] and most recently R-subunit bound to the C-subunit [Kim, C., et al., (2005), Crystal structure of a complex between the catalytic and regulatory (RIα) subunits of PKA, Science 307, 690-696] allow correlation between sequence and structure. The R-subunit is composed of two homologous domains with one cAMP binding site each (named A and B sites) residing in each domain. Although both A and B sites bind cAMP with high affinity, their structural and sequence (FIG. 1) divergence suggests that small molecules can be discovered that distinguish between them. Indeed, synthetic cAMP analogs have been designed that bind preferentially to one site [Schwede, F., et al., (2000), Cyclic nucleotide analogs as biochemical tools and prospective drugs, Pharmacol Ther 87, 199-226].
There is also sufficient sequence divergence between RI and RII isoforms to suggest that selective binders may be designed. Inspection of the residues directly in contact with cAMP (FIG. 1) shows that 6 out of 19 residues in the A site and 11 out of 22 residues in the B site differ between types I and II. The discovery of isoform specific R-binders then opens up the possibility for targeting only the disease associated forms of PKA.
Therefore, there is a need for an assay system, amenable to high throughput screening, to detect small molecule agonists or antagonists for the Iα PKA holoenzyme.